The number of protein types present in human body is approximately 100,000. Function of the protein is finely regulated by various post-translational modifications including cleavage by various protease, regulation of the activity or interaction by addition of a carbohydrate moiety, phosphate group, and the like, and localization to membrane by acylation such as myristylation and palmitilation. In particular, in the case of eukaryote, it is rather rare that the proteins synthesized based on the gene arrangement function without further regulation, and they are usually modified in various ways after the synthesis on the ribosome at the site of its synthesis or in various stages before the final localization in the cell is determined. The biopolymer undergoing such spatiotemporal changes can not be identified solely by the genomic information, and the identification will be enabled only after conducing direct analysis of the protein.
One technique used for such structural analysis is mass spectroscopy. Use of such mass spectroscopy has enabled to obtain information on the sequence and the post-translational modification of polypeptides (peptides or proteins comprising amino acid molecules connected by peptide linkage) constituting the biopolymer. In particular, ion trap mass spectroscopy using a radio frequency electric field, mass spectroscopy using a quadrupole mass filter, and time-of-flight (TOF) mass spectroscopy exhibiting high throughput are highly compatible with the pretreatment means such as liquid chromatography system used for the sample separation. Accordingly, these methods are highly appropriate in proteome analysis in which a large variety of samples are continuously analyzed, and these methods are widely used in such application.
In mass spectrometry, sample molecules are generally ionized and introduced in a vacuum (or ionized in a vacuum), and motion of the resulting ion in an electromagnetic field is measured to thereby determine mass-to-charge ratio (m/z) of the target molecule ion. Since the obtainable information is the mass-to-charge ratio which is a macroscopic quantity, information on the internal structure can not be obtained by only one mass spectroscopy operation, and therefore, a method called “tandem mass spectrometry” is used. In the tandem mass spectrometry, the sample molecule ions are isolated and isolated in the first mass spectroscopy operation, and these ions are referred to as precursor ions. The precursor ions are then dissociated by some means, and the dissociated ions are referred to as fragment ions. The fragment ions are then subjected to mass spectroscopy to thereby obtain information on the patterns of the fragment ions. Since each dissociation method has its own dissociation pattern, the sequence structure of the precursor ion can be estimated by using such dissociation pattern. In particular, in the field of analyzing biological molecules having amino acid backbone, dissociation is carried out by such means as collision induced dissociation (CID), infra red multi photon dissociation (IRMPD), electron capture dissociation (ECD), or electron transfer dissociation (ETD).
The technique currently most widely used in the field of protein analysis is CID. In the CID, the precursor ions are kinetically energized and collided with a gas, and the molecular vibrations of the precursor ions are excited by this collision so that dissociation occurs at the weak sites of the molecular chain. A method which has recently come into use is IRMPD. In the IRMPD, the precursor ion is irradiated with infrared laser beam, and the precursor ions are allowed to absorb a large number of photons. This excites molecular vibrations, and the dissociation occurs at the weak sites of the molecular chain. The sites of dissociation are shown in FIG. 11, where a, b, and c respectively designate molecules including the moiety on their NH2 terminal side, and x, y, z respectively designate molecules including the moiety on their COOH terminal side. The weak sites from which dissociation may occur in the CID or IRMPD are the sites “a-x” and “b-y” in the backbone of the molecule comprising the amino acid sequence. However, depending on the pattern of the amino acid sequence, some of the “a-x” and “b-y” sites are less likely to become cleaved, and it is known that complete structural analysis cannot be accomplished only by the CID or IRMPD. In such a case, pretreatments using an enzyme or the like would be required, and this is the main impediment in realizing the high speed mass spectroscopy. In the case of biomolecules which have undergone post-translational modification, the polypeptide side chain added by the post-translational modification tends to become cleaved in the course of the CID or the IRMPD. Because of this cleavage of the side chain, type of the modified molecule and presence/absence of the modification can be estimated from the lost mass. However, the critical information on the site (i.e., the amino acid residue) of the modification would be lost.
On the other hand, in the case of ECD (electron capture dissociation) and ETD (electron transfer dissociation), cleavage site does depend on the amino acid sequence, and the backbone of the amino acid sequence is cleaved at one position, namely, at c-z site in FIG. 11 (with the exception of cyclic structures in which proline residue is not cleaved). Because of this, the protein molecules can be completely analyzed solely by means of mass spectroscopy technique. In addition, ECD and ETD are suitable for use in the study and analysis of post-translational modification since the side chain is less likely to be cleaved in the ECD and ETD. As a consequence, ECD and ETD have recently received particular attention as promising dissociation techniques.
However, ECD and ETD are capable of dissociating only positively charged multivalent ions. ECD is based on the principle of molecular dissociation by the capturing of the negatively charged electron by the positively charged molecule. Therefore, dissociation is not accomplished in the case of the negatively charged molecule, and also, the positively charged monovalent ions are not detected in the mass spectrometer system since such ion becomes a neutral molecule.
In the case of ECD, reaction efficiency is said to be proportional to the square of the electric charge, and therefore, protein can be efficiently dissociated since the protein has an extremely high valence. Because of this, an analysis called “top down analysis” has become a focus of attention. In this top down analysis, a protein is directly subjected to mass spectroscopy, as opposed to the “bottom up analysis” in which the protein is preliminarily digested by enzymes for its fragmentation before the mass spectroscopy. While the bottom up analysis has been unable to identify whether the analyte peptide is present in the form of a protein or in digested form in the living body, such identification has been enabled by the top down analysis since the biological sample is directly analyzed.
ECD is currently realized by Fourier-transform ion cyclotron resonance (FT-ICR) mass spectroscopy and radio frequency ion trap mass spectroscopy (Takeshi Baba et al. Electron Capture Dissociation in a Radio Frequency Ion Trap. Anal. Chem. 2004, Aug. 1; 76(15): 4263-6). FT-ICR has realized a resolution at the level of about 800,000 and an exact mass measurement at the level of ppb. FT-ICR, however, requires a strong parallel magnetic field of at least several tesla through the use of a superconducting magnet, and therefore, it is expensive and large-sized. FT-ICR also requires a measurement time of about several to 10 seconds for obtaining the data for one spectrum, and another about 10 seconds for the Fourier analysis necessary for obtaining the spectrum. This means that a total period of several dozen seconds is required for one spectrum in FT-ICR, and compatibility with liquid chromatography with the peak appearing in a time period of about 10 seconds is far from sufficient. On the other hand, when the radio frequency ion trap is combined with TOF, the data can be measured at the high resolution of about 15,000 and the exact mass measurement at the level of ppm, which may not be as high and exact as the FT-ICR. However, this combination is inexpensive and small-sized, and since this combination is capable of conducting the measurement at a high speed, it is highly compatible with the liquid chromatography (Hiroyuki Satake et al., Anal. Chem. 2007, 79, 8755-8761).
As described above, large scale protein analysis is being implemented by the progress of the mass spectrometer system and the liquid chromatography as well as the improvement of database technology. However, the number of proteins that can be analyzed in one cycle is yet several thousands at the very best, and this is why “focused proteome” not subjecting all but a particular set of the protein is currently the mainstream of the protein analysis.
For example, analysis focusing on phosphorylation which is a typical post-translational modification is widely practiced. However, analysis of the phosphorylation has two problems to be overcome. First, phosphorylation product may exhibit an ionization efficiency that is several hundred to several thousand times lower than that of the non-phosphorylation product. Second, when CID is used for the dissociation, proteins should be preliminarily decomposed into peptide fragments by enzymatic digestion before the CID since CID is incapable of dissociating the protein. For example, it has been estimated that, if a protein having a molecular weight of 40,000 is cleaved to produce 400 peptide fragments by enzymatic digestion, the phosphorylation product that had constituted about 30% of the entire protein would be reduced to the level of 0.08% or less by the enzymatic digestion (Experimental Medicine, Vol. 23 No. 19 2005, pp 2951-2956, in Japanese).
Because of these two limitations, phosphorylated peptides will rarely be identified if peptide fragments of the biological sample were subjected to mass spectroscopy with no further treatment. Accordingly, a technique utilizing the leaving of the post-translational modification site which is characteristic to the CID is used as a common technique to enable specific detection of the phosphorylated peptide. In the tandem mass spectroscopy using CID, a fragment ion comprising the phosphorylated peptide from which only the phosphate group has been lost and a fragment ion derived from the phosphate group are generated. This method for searching the phosphorylated peptide by focusing on the phosphate group is called “precursor ion scanning”.
This precursor ion scanning is a technique in which all precursor ions generating a particular fragment ion are scanned. The ions that had passed through the scanning in the first mass separator MS-1 is subjected to CID in the collision cell, and of the resulting fragment ions, the ions having a particular m/z are detected in the second mass separator MS-2. When the particular fragment ions are detected in the MS-2, corresponding precursor ion that had passed through the MS-1 is identified. When the phosphate group is specifically detected, the ion having an m/z of 79 (PO3−) is detected in negative mode (see “Experimental Protocols in Proteomics (in Japanese)” Shujun-sha, pp. 156-168).